Fluorescence anisotropy analysis of EphB2 clusters at contacts with ephrinB2-expressing cells. Nonuniform clustering response of kdEphB2 upon contact with ephrinB2-expressing cells. COS-7 cells transiently transfected with kdEphB2-mGFP (A) were co-cultured with HEK293 cells stably expressing wild-type mCherry-ephrinB2 (B; merged image in C). Homo-FRET between kdEphB2-mGFP was determined by fluorescence anisotropy (D). Representative experiment from a series of co-cultures (n = 20 cells analyzed). (E) For calibration of anisotropy values to cluster size distributions the cumulative relative contributions of 0FKBP (monomer, n = 20 cells), 1FKBP (dimer, n = 22 cells), and 3FKBP (oligomer, n = 57cells) from n = 3 independent experiments were plotted to the range of anisotropy values on the x-axis (see Materials and methods). (F) Areas with color coded and calibrated anisotropy pixel distributions of predominantly monomers, dimers, and multimers according to the histogram (G). Contact areas in which the anisotropy values were too low to be quantified (because of intensity saturation) are shown in black pixels. Bars, 10 µm.